: The goal of this research is to elucidate the role that cytochrome P450 2D6 variants may play in neurotoxicity and neurodegenerative diseases. Polymorphism in cytochrome P450 2D6 (P450 2D6), an enzyme that has been identified in human brain and known to play a role in the metabolism of both foreign and endogenous pro-neurotoxins has been proposed to have an association with Parkinson's disease. It is our goal to move the understanding of the relationship between P450 2D6 polymorphism and Parkinson's disease to the molecular level and, ultimately, to provide a mechanistic basis for the complex interplay of genetic and environmental factors in the etiology of this disease. Although this application focuses on the evaluation of a single P450 2D6 mutant, which exhibits an Arg296 to Sys296 mutation and has the highest risk factor yet identified for the disease (=5.56), the protocol developed will be generally applicable to the investigation of the roles of any P450 variant(s) in neurotoxicity. Specifically, we propose to produce P450 2D6 and its variant in sufficient quantity for in vitro investigations by over expression in COS and Sf9 cells. We will define the in vitro structure-function relationships between the wild type and mutant P450 2D6 enzymes with regard to: (1) substrate metabolism and/or enzyme inhibition; and (2) the generation of reactive intermediates or reactive oxygen species or a family of structurally related tetrahydro-isoquinoline, b-carboline, and pyridine compounds, that are established or proposed to contribute to the etiology of Parkinson's disease. Finally, we propose to transfect a catecholaminergic cell line, rat pheochromocytoma PC12 cells, with the wild type and mutant enzymes. Differential sensitivities between the transfectants with regard to markers for neuronal stress and toxicity upon treatment with the potential pro-neurotoxins identified through our in vitro studies may illustrate the cellular consequences of such metabolic differences. With validation, these and analogously transfected neuronal cell lines may enable the development of assays for assessing the neurotoxicity of endogenous and foreign compounds that are responsible to cerebral metabolism by P450 2D6.